Thalidomide and its analogs lenalidomide and pomalidomide (IMiDs), are highly effective treatments for hematologic malignancies such as multiple myeloma (MM) or del(5q) myelodysplastic syndrome (5q-MDS), as well as an FDA approved treatment for erythema nodosom leprosum (ENL). Over decades IMiDs were found to have broad but not necessary related effects, ranging from induction of oxidative stress, inhibition of angiogenesis, to multiple effects on the immune system such as enhanced production of cytokine interleukin-2 (IL-2) and inhibition of cytokine tumor necrosis factor (TNF). A seminal breakthrough in understanding the activity of IMiDs was the discovery that IMiDs bind CRBN, the substrate receptor of the CUL4-RBX1-DDB1- CRBN (CRL4CRBN) E3 ubiquitin ligase, and to exhibit dual activity: 1) preventing CRL4CRBN from ubiquitinating its native substrates, including MEIS2, and 2) to alter the specificity of the CRL4CRBN ubiquitin ligase to ubiquitinate new targets, notably the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3), and casein kinase 1 alpha (CK1?). Therefore IMiDs impart gain-of-function properties to the CRL4CRBN substrate receptor that enable binding and ubiquitination of key therapeutic targets. While IKZF1/3 and Ck1? provide a plausible explanation for IMiD efficacy, they fall short in explaining all of the cellular and clinical response induced by IMiDs and other substrates likely exist. Moreover, the finding that IMiDs act by altering the substrate repertoire of the CRL4CRBN ubiquitin ligase towards neo-substrates has transformative potential to drug discovery; however, we do not understand the molecular basis of neo-substrate recruitment. Important unresolved issues are 1) the complete substrate repertoire of IMiDs, 2) the structural features of the degron recognized by a CRL4CRBN-IMiD complex, and 3) how on molecular grounds, modifications to IMiDs can confer substrate selectivity. In Aim 1 we will validate a list of high-confidence substrate candidates generated with a novel pulse-SILAC mass spectrometry approach. We will publish validated substrates as a resource to the community, which will greatly facilitate the in-depth understanding of the clinical and cellular outcomes of IMiD treatment. In Aim 2, we will use a combination of X-ray crystallography, biochemical reconstitutions and cellular experiments to address the molecular basis of IMiD activity. In depth understanding of this novel drug mechanism will guide development of future medicine. Leveraging our intricate molecular and structural understanding, we will develop IMiD derivatives with altered substrate selectivity in Aim 3 of this proposal. Through a set of quantitative assays developed in the lab, we are for the first time able to follow a structure and mechanism guided approach to synthesize derivatives of IMiDs and explain how subtle chemical modifications result in altered substrate specificity. Taken together, we will define the molecular framework that underlies IMiD induced ligase repurposing.